Cytokines released by both T-lymphocytes and activated macrophages have been implicated as immunological effector molecules that both inhibit insulin secretion from the pancreatic beta-cell and induce beta-cell destruction. Our studies have demonstrated that production of the free radical nitric oxide (NO), resulting from the expression of the inducible isoform of NO synthase (iNOS), mediates these deleterious effects. The cellular mechanism responsible for inhibition of beta-cell function and destruction by NO involves, in part, inactivation of enzymes specifically localized to the beta-cell mitochondria that contain iron-sulfur centers or clusters. Our recent studies indicate that IL-1 induces the co- expression of both iNOS and the cytokine-inducible isoform of cyclooxygenase, COX-2. The expression of COX-2 results in the overproduction of the proinflammatory prostaglandins (PGE2) and thromboxanes. We have also recently demonstrated that NO directly stimulates the activities of both constitutive and inducible isoforms of COX further augmenting the overproduction of these proinflammatory mediators that may be important in initiating or maintaining the inflammatory response associated with autoimmune diabetes. In specific aim 1, we will characterize by both biochemical and molecular approaches the role of tyrosine kinase(s) and the transcriptional factors NFkappaB, c-Fos and c-Jun in the signal transduction pathway for IL-1-induced co- expression of iNOS and COX-2 by islets and primary beta-cells. This information will be utilized in developing therapeutic strategies that may selectively block overproduction of NO and other inflammatory mediators. Cytokines released by infiltrating immune cells have also been implicated in autoimmune diabetes by recruitment of lymphocytes and macrophages to the site of inflammation. In specific aim 2, we will identify and characterize compound(s) released by islets exposed to cytokines or LPS into the culture media including monocyte chemoattractant protein-1 (MCP- 1) that mediate specific chemotaxis for monocytes and macrophages and contribute to the inflammation associated with autoimmune diabetes. Our recent studies also indicate that activation of intraislet macrophages results in endogenous release of IL-1 that attains sufficient local concentrations within islets to induce iNOS and COX-2 protein expression in beta-cells. In specific aim 3, we have developed a bioassay for IL-1 release and made the important findings that the release of IL-1 from a macrophage cell line, RAW 264.7 and peritoneal macrophages is dependent on the concomitant production of NO. In this aim, we will determine if NO- dependent IL-1 release from macrophages is mediated by a direct effect of NO on IL-1 precursor processing via IL-1beta concerting enzyme (ICE) or by NO-mediated apoptosis. In specific aim 4, we will employ selective inhibitors of iNOS and COX-2 to prevent pancreatic islet destruction in the low-dose STZ model of diabetes and in the development of immunologically-mediated diabetes in NOD mice. Our previous findings indicate that NO is produced by isolated islets from recipient male NOD mice following adoptive transfer of spleen cells from diabetic female NOD mice and suggest a NO-sensitive and insensitive phase in this animal model of diabetes. Additional strategies of intervention that also block iNOS and COX-2 protein expression as identified in specific aim 1 of this proposal will also be employed. This comprehensive approach will allow evaluation of the role(s) of NO and proinflammatory prostaglandins in the development of autoimmune diabetes.